KR102114107B1 - Pharmaceutical composition for treating cancer comprising exosome like vesicle derived from natural killer cell - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for improving or treating cancer comprising an exosome-like vesicle derived from natural killer cells as an active ingredient, wherein the vesicle can effectively suppress proliferation by targeting cancer cells.

Description

Pharmaceutical composition for treating cancer comprising exosome like vesicle derived from natural killer cell}

The present invention relates to a pharmaceutical composition for improving or treating cancer, comprising as an active ingredient an exosome-like vesicle derived from natural killer cells.

Natural killer cells (NK cells) are one of the innate immune cells, and are cells that show selective cytotoxicity against cancer cells. Natural killer cells, unlike other immune cells, can detect and immediately remove cancer cells because they can differentiate cancer cells from normal cells through various immune receptors on the surface of natural killer cells. In many cancer patients, defects are found in the number of natural killer cells or anticancer activity, and it is known that dysfunction of natural killer cells is closely related to the occurrence of these cancers.

The process by which natural killer cells kill cells involves the release of cytoplasmic granules, including perforin and granzyme, and the process including FasL and TRAIL. Natural killer cells secrete various cytokines such as IFN-γ, INF-α, GM-CSF and IL-10, and express several receptors on the cell surface, which are cell adsorption, activation of cell killing capacity, or cells It is involved in the suppression of killing ability.

On the other hand, exosomes (exosomes) are nano-sized vesicles that are naturally made in cells, and contain protein and genetic information, so they transmit various signals, including genetic information, out of the cells to other cells to develop, proliferate, and differentiate. , Immune regulation, angiogenesis, progression of various diseases, etc. In the case of exosomes that are biological nanovesicles, the immune response can be avoided, human body compatibility is excellent, and drug delivery function, target delivery effect to specific cells, stability in blood, etc. I am receiving. However, naturally produced exosomes have a very small amount, and the separation and purification process is complicated.

The present inventors completed the present invention by preparing vesicles derived from exosomes that are easier to produce than exosomes and have anti-cancer effects as a result of research to improve the disadvantages of the exosomes.

1. Republic of Korea Registered Patent No. 10-1762833

An object of the present invention is to provide a pharmaceutical composition for improving or treating cancer and a contrast agent for diagnosing cancer, comprising as an active ingredient an exosome-like vesicle derived from natural killer cells.

In order to achieve the above object, an aspect of the present invention provides a pharmaceutical composition for improving or treating cancer, which includes an exosome-like vesicle derived from natural killer cells as an active ingredient.

As used herein, the term 'exosome like vesicle' refers to a membrane-structured vesicle that performs a function similar to that of exosomes secreted from natural killer cells. It is classified and contains the cell membrane lipid, membrane protein and cytoplasmic components of natural killer cells.

According to one embodiment of the present invention, the exosome-like vesicle is derived from natural killer cells and can separate significantly more exosomes secreted from natural killer cells.

According to one embodiment of the present invention, the exosome-like vesicle may have a size of 50 to 200 nm, and may specifically have a size of 80 to 140 nm.

According to one embodiment of the present invention, the exosome-like vesicle induces apoptosis of cancer cells and inhibits tumor growth, and thus can be usefully used as a pharmaceutical composition for improving or treating cancer.

The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceuticals in addition to including exosome-like vesicles as active ingredients.

The pharmaceutical composition according to the present invention is formulated in the form of oral dosage forms, external preparations, suppositories and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. Can be used. Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the composition of the present invention, such as starch, calcium carbonate, sucrose It is prepared by mixing (sucrose) or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweetening agents, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerol gelatin may be used.

The preferred dosage of the composition of the present invention depends on the patient's condition and body weight, the degree of disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. The administration may be administered once a day, or may be divided and administered several times, and the above dosage does not limit the scope of the present invention in any way.

Another aspect of the present invention provides a contrast agent for diagnosing cancer that includes exosome-like vesicles derived from natural killer cells as an active ingredient.

As used herein, the term 'contrast agent' is a substance used to artificially make a difference in contrast and to display it as an image for the purpose of diagnosis.

According to one embodiment of the present invention, since the exosome-like vesicle is absorbed by cancer cells, the exosome-like vesicle and a fluorescent probe, nanoparticles, etc. are combined to be usefully used as a contrast agent to check whether cancer has developed or not. Can be.

Another aspect of the present invention comprises the steps of: extruding the natural killer cell suspension into a membrane filter; 초원 ultracentrifuging the extruded suspension using an iodixanol density gradient; And ⒞ resuspending the centrifuged pellet, to provide a method for isolating exosome-like vesicles derived from natural killer cells.

According to one embodiment of the present invention, the step ⒜ may be performed by extruding the natural killer cell suspension into polycarbonate membrane filters having a size of 0.5 μm to 7 μm. For example, the natural killer cell suspension can be sequentially extruded 8 times into 5 μm and 1 μm polycarbonate membrane filters. By sequential extrusion, natural killer cells are gradually divided into small-sized vesicles, and finally exosome-derived vesicles can be obtained.

According to one embodiment of the invention, the ultracentrifugation of the step ⒝ can be carried out for 1 hour to 6 hours at 100,000g to 160,000g, specifically for 4 hours at 132,000g.

According to an embodiment of the present invention, exosome-like vesicles derived from natural killer cells can be effectively used to improve or treat cancer because they can effectively suppress proliferation by targeting cancer cells.

Figure 1 shows the results of confirming the expression of the effluc gene in D54 / F cells in which the effluc gene is introduced into a human glioblastoma cell line.
2 shows the results of measuring the activity of luciferase in D54 / F cells.
3 schematically shows a method for isolating exosomes (NK-Exo) or exosome analogs (NK-EM) from natural killer cells.
4 shows the results of comparing proteins expressed in exosomes and exosome analogs.
Figure 5 shows the results of comparing the number of particles and the amount of protein contained in the exosomes and exosome analogs.
Figure 6 shows the results of the analysis of the form of exosomes and exosome analogs by projection electron microscopy and nanoparticle tracking.
7 shows the results of confirming whether cytotoxicity of exosomes and exosome analogs to D54 / F cells.
Figure 8 shows the results confirming the cytotoxicity of exosomes and exosome analogues in MDA-MB-231 / F, HepG2 / F and CAL-62 / F cells into which the effluc gene has been introduced.
9 shows the results of confirming the cytotoxic level of the exosome analogs according to the treatment concentration in D54 / F cells.
Figure 10 shows the results confirming the cytotoxic level of the exosome analogs according to the treatment concentration in MDA-MB-231 / F, HepG2 / F and CAL-62 / F cells.
11 shows the results confirming the degree of absorption of exosomes and exosome analogs by D54 / F cells.
12 shows the results confirming the expression levels of FasL (Fas ligand) and perforin in exosomes and exosome analogs.
Figure 13 shows the results confirming the cytotoxic level of the exosome analogues after treatment with FasL inhibitors in D54 / F cells.
FIG. 14 shows the results of confirming the expression level of a protein related to apoptosis after treating an exosome analog to D54 / F cells.
Figure 15 shows the results of confirming the degree of in vivo distribution of exosome analogs by injecting D54 / F cells into mice to induce tumor development and then administering exosome analogs.
Figure 16 shows the results of confirming the degree of absorption of the exosome analogues by administering an exosome analogue to a mouse inducing tumor development and separating organs.
FIG. 17 shows the results of administering exosome analogs to mice inducing tumor development, and separating organs to graphically display the degree of absorption of exosome analogs.
Figure 18 shows the results of confirming the degree of tumor growth over time after administration of exosome analogues to mice inducing tumor development.
Figure 19 shows the results of graphically showing the degree of tumor growth over time after administration of exosome analogues to mice inducing tumor development.
20 shows the results of confirming the degree of absorption of exosome analogs by separating organs after administration of the exosome analogs to mice inducing tumor development.
FIG. 21 shows the results of confirming the tumor size and weight by ultrasound imaging after administering an exosome analog to a mouse inducing tumor development.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Experiment method

1. Cell culture

The human natural killer cell (hereinafter referred to as NK cell) cell line NK92-MI was purchased from ATCC (American Type Culture Collection, Rockville, MD, USA). NK92-MI is 2% exosome-depleted human serum (exosomes-depleted humanserum; ultracentrifugation at 120,000g for 18 hours) and 1% penicillin-streptomycin (Hyclone, Logan, UT, USA) added stem cell culture Incubated with medium (Cellgro, Freiburg, Germany) at 37 ° C. and 5% CO ₂ conditions. Exosomal-deficient human serum was prepared by ultracentrifuging human serum at 120,000xg for 18 hours. Human glioblastoma cell line D54, breast cancer cell line MDA-MB-231, undifferentiated thyroid cancer cell line CAL-62 and liver cancer cell line HepG2 are 10% fetal bovine serum (FBS; Gibco) and 1% Cultured in DMEM medium (Dulbecco's modified Eagle's medium; Hyclone, Logan, UT, USA) to which penicillin-streptomycin was added.

The D54, MDA-MB-231, CAL-62 and HepG2 cell lines were transformed with a recombinant retrovirus containing a plasmid to prepare a transformed cell line. The plasmid includes an enhanced firefly luciferase (effluc) gene and Thy1.1 gene, which are activated by a long terminal repeat promoter (LTR promoter). Thy1.1 positive cells were sorted by magnetic cell sorting (Magnetic-activated cell sorting, MACS; Miltenyi Biotec, Auburn, CA, USA) to isolate cells expressing both the effluc and Thy1.1 genes. After separating the double positive cells for the Effluc and Thy1.1 genes, RT-PCR (reverse transcription polymerase chain reaction) and Western blot were performed to confirm whether the effluc gene was expressed at the mRNA and protein levels.

As a result, as shown in FIG. 1, it was confirmed that the effluc gene was expressed in D54 cells into which Effluc and Thy1.1 genes were introduced. In FIG. 1, A is a Western blot result, and B is an RT-PCR result.

Hereinafter, the transformed cell line is described as the cell line name / F, such as D54 / F.

In addition, in order to confirm the luciferase activity, D54 and D54 / F were cultured by inoculating black or transparent bottom 96-well plates at various densities (0, 0.25 x 10 ⁴ , 0.5 x 10 ⁴ , 1 x 10 ⁴ and 2 x 10 ⁴ ). . After 24 hours, 3 μl of D-luciferin (D-luciferin; 3 mg / ml) was added to each well, and the activity of luciferase was measured with an IVIS LuminaIII imaging system (Perkin-Elmer, Wellesley, MA, USA). .

As a result of the measurement, it was found that the fluorescence signal increases in proportion to the cell density, as shown in FIG. 2, which means that luciferase works normally in D54 / F cells.

2. Exosome ( exosome ) And Exosome  Analogue ( exosome mimetics )

The NK92-MI cell (size 7 μm) suspension was made using a mini-extruder (Avanti Polar Lipids, Birmingham, AL) with 5 μm and 1 μm size polycarbonate membrane filters; Nuclepore, Whatman, Inc. ., Clifton, NJ) were sequentially extruded 8 times. 2 ml of 10% and 60% iodixanol (Otidixanol; OptiPrep ?, Sigma-Aldrich, USA) was added to the bottom of the ultracentrifuge tube (Beckman Coulter, Brea, CA, USA), respectively. 1 ml of the suspension sample was added to the tube, followed by ultracentrifugation (132,000 g and 4 ° C.) for 4 hours. The exosome analogues are located in the boundary layer between 10% iodixanol and 60% iodixanol, and then separated and further centrifuged (100,000xg and 4 ° C) for 1 hour to obtain exosome analog pellets. The obtained pellet was filtered through a 0.22 μm filter, resuspended in PBS, and used in the experiment while storing at -80 ° C. In addition, exosomes were isolated from NK cells according to methods known in the art. Hereinafter, exosome analogs are described as NK-EM, exosomes derived from NK cells are described as NK-Exo, and NK-Exo was used as a control in in vitro experiments.

3 schematically shows a method of isolating exosomes (NK-Exo) or exosome analogs (NK-EM) from NK cells.

3. Exosome  Analogs and Exosome  analysis

The size, distribution and number of particles for Exosomal formulations were analyzed at 25 ° C. with a NanoSight LM10 system (Nanosight Ltd., Amesbury, UK) equipped with a 405 nm laser. To this end, NK-EM and NK-Exo were prepared at a concentration of 0.01 mg / ml and analyzed in a device equipped with Nanoparticle Tracking Analysis (NTA) 2.0 analysis software. In addition, in order to compare the production amount of NK-EM and NK-Exo, ⁶ NK cells 5x10 ⁶ were prepared to simultaneously separate NK-EM and NK-Exo, and the total number of proteins and nanoparticles was confirmed. All experiments were diluted 1: 1000 to proceed to maintain the particle concentration of 10x10 ⁷ / mL, and repeated experiments three times.

4. Western Blotting (Western blotting)

Western blotting was performed according to the following method known in the art. Whole cell lysates, NK-EM and NK-Exo are prepared in RIPA buffer (Thermo Scientific), and 50 μg / well protein is separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamidegel electrophoresis) Did. Subsequently, the protein was transferred to a PVDF membrane (polyvinylidene difluoride membrane; Millipore), and sequentially reacted with a primary antibody and a secondary antibody linked with horseradish peroxidase (HRP). Protein bands were confirmed according to the manufacturer's protocol with an enhanced chemiluminescence (ELC) detection system (GE healthcare, Milwaukee, WI, USA). Primary antibodies used were as follows: CD63, GM130, Alix, cytochrome-c (Abcam), perforin and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Also, Mem-PER? After FasL was separated with Plus Membrane Protein Extraction Kit (ThermoFisher Scientific, Waltham, MA, USA), it was confirmed by Western blotting.

In addition, Changes in the expression of proteins involved in cell death and cell signaling pathways were confirmed in tumor cells treated with NK-EM. The primary antibodies used were: caspase-3, cleaved caspase-3, cleaved (Poly (ADP-ribose) polymerase), cleaved PARP), Cytochrome-C (cytochrome-C), phosphorylated extracellular signal-regulated kinases (ERK), phosphorylated ERK (p-ERK), phosphorylated protein kinase B (AKT) and phosphorylated AKT (p-AKT).

5. Transmission Electron Microscopy, TEM ) observe

The separated NK-EM and NK-Exo were contacted with glow-discharged carbon-coated copper grids (Electron Microscopy Sciences, Fort Washington, PA), followed by 20 minutes in a dry environment at room temperature. Adsorbed. The grid was fixed with 2% paraformaldehyde (PFA) for 10 minutes, and washed by dropping deionized water. In order to remove the negative background, the grid was stained by reacting 1% glutaraldehyde for 15 minutes. Electron micrographs were recorded with an HT 7700 transmission electron microscope (Hitachi, Tokyo, Japan).

6. NK - Exo  And NK - EM  Cell toxicity cytotoxicity ) Check whether

6-1. BLI (bioluminescence imaging)

D54 / F cells were cultured in 96-well black plates using serum-free medium, and treated with NK-EM or NK-Exo at concentrations of 10, 20, and 30 μg, followed by different times (24, 48, and 72 hours). Cultured for a while. Thereafter, the absorbance was measured at 450 nm using a microplate reader. BLI was performed after contacting MDA-MB-231 / F, CAL-62 / F and HepG2 cells with different concentrations of NK-EM or NK-Exo for 72 hours in the same manner as above.

6-2. CCK -8 assay

D54 / F cells were cultured in 96-well black plates, and treated with NK-EM or NK-Exo at concentrations of 10, 20 and 30 μg and then cultured at different times (24, 48 and 72 hours). Thereafter, 10 µl of a CCK-8 (Dojindo Molecular Technologies, Tokyo, Japan) solution was added to each well and further cultured in a humidified incubator for 2 hours. Absorbance was measured at 450 nm with a microplate reader. In addition, after contacting MDA-MB-231 / F, CAL-62 / F and HepG2 cells with different concentrations of NK-EM or NK-Exo for 72 hours, CCK-8 was performed in the same manner as above.

7. NK - Exo  And NK - EM  Cellular uptake assay

Dispense NK-EM and NK-Exo suspensions onto the plate, fluorescent lipophilic tracer 2- (5- (1,3-dihydro-3, 3-dimethyl-1-octadecyl-2H-indol-2-ylidene) -1, 3-pentadienyl) -3, 3-dimethyl-1-octadecyl-perchlorate (Invitrogen, Carlsbad, CA, USA; hereinafter referred to as DiD) was added and reacted at room temperature for 20 minutes. Thereafter, the plate was washed three times with PBS, and after removing the supernatant, DiD-labeled NK-EM and DiD-labeled NK-Exo (hereinafter referred to as DiD-NK-EM and DiD-NK-Exo, respectively) were PBS. Resuspended in. 30 μg of DiD-NK-EM or DiD-NK-Exo was reacted with D54 / F cells at 37 ° C. for 3 hours. After 3 hours D54 / F cells were washed 5 times with PBS and fixed with 4% PFA for 20 minutes. A Vectashield Mounting solution (Vector Laboratories, Burlingame, CA, USA) containing DAPI (4 ′, 6-diamidino-2-phenylindol) is immobilized on immobilized cells, and a confocal laser microscopy; Zeiss, LSM 800 with Airyscan, Germany).

8. In the tumor mouse model NK - EM  Check distribution and tumor target

Experimental animals use 6-week-old female SFP (Specific Pathogen-free) BALB / c nude mice (Hamamatsu, Shizuoka, Japan), and the heterologous tumor mouse model is a known method (Theranostics. 2017; 7 (10): 2732). -2745).

Briefly, 5 × 10 ⁶ D54 / F cells were injected subcutaneously in the mouse to induce tumor growth for 28 days, and DiD-NK-EM (total protein amount 100 μg) or PBS (phosphated buffered saline) was used for the tail blood vessels of the mouse. Was injected into. Fluorescence lifetime imaging (FLI) of DiD-NK-EM was confirmed by an in vivo imaging system (IBIS). The auto-fluorescence background was removed using an imaging control mouse without NK-EM injection. After 24 hours, organs (spleen, liver, kidney, lung, and heart) and tumor tissues were recovered from mice to perform ex vivo FLI, and images were quantified.

9. NK - EM  Confirmation of anti-tumor effect in vivo

D54 / F cells were injected subcutaneously into mice and divided into two groups (n = 5 / group) of the experimental group and the control group at random 10 days after injection. Did-NK-EM 30 μg or PBS was injected into the experimental group and control group 3 times every 3 days intratumorally. Tumor BLI was confirmed on days 0, 4, 9, 14 and 19 and 24 after administration of the Did-NK-EM suspension, and ultrasound 3-D imaging (S-Sharp Corporation, New Taipei City, Taiwan) The volume of the tumor was measured. On the 24th day after administration of the Did-NK-EM suspension, mice were sacrificed to confirm X-Vivo BLI and tumor BLI, and tumor weight was measured.

10. Statistical Analysis

All experimental results were described as mean ± standard deviation, and inter-group differences were tested by two-tailed Student's t-test. When the P value was <0.05, it was judged as having statistical significance.

Experiment result

One. NK - EM  Character analysis

As a result of Western blotting performed on NK-Exo and NK-EM isolated from NK cells, CD63 and ALIX [ALG-2 (apoptosis-linked gene 2) -interacting protein X in NK-EM as shown in FIG. 4. ] Was confirmed to contain two types of protein. In addition, unlike NK-Exo, NK-EM is rich in cellular proteins such as GM-130 (cis-Golgi matrix protein of 130 kD), cytochrome-C and beta-actin. It was confirmed that it exists.

As a result of separating NK-EM and NK-Exo simultaneously using ⁶ NK cells 5x10, as shown in Fig. 5A, NK-EM contains 18.68 μg of protein and NK-Exo 1.36 μg of protein. I could confirm that. As a result of comparing the number of generated particles, as shown in B of FIG. 5, 9.59x10 ⁷ NK-EMs were generated, ⁶ NK-Exo 1.86x10s were generated, and NK-EMs were generated approximately 50 times more. I could confirm. In FIG. 5, *** represents p <0.001.

As a result of confirming the separated NK-Exo and NK-EM by TEM, it was found that both NK-Exo and NK-EM are spherical particles having a size of 100 to 150 nm having a complete membrane structure, as shown in FIG. 6A. In addition, as a result of nanoparticle tracking analysis (Nanoparticle tracking analysis, NTA), it was confirmed that NK-EM has a size in the range of 99.2 ± 21.5 nm and NK-Exo has a size in the range of 118 ± 33.1 nm, as shown in B of FIG. 6. Could.

2. NK - EM  Check for cytotoxicity

D54 / F, MDA-MB-231 / F, CAL-62 / F and HepG2 / F cells were incubated with different concentrations of NK-EM or NK-Exo, followed by BLI and CCK-8 assays. .

As a result of the experiment, as shown in FIG. 7, it was confirmed that the BLI signal intensity decreased as the treatment concentration and treatment time of the NK-EM increased as the D54 / F cells decreased, thereby decreasing the cell viability. In addition, it was confirmed that the cell viability was significantly lower in the experimental group treated with NK-EM for 48 hours and 72 hours compared to NK-Exo. In FIG. 7, * denotes p <0.05, ** denotes p <0.01 and *** denotes p <0.001, ## denotes p <0.01 and ### denotes p <0.001, and is the same in the following drawings.

In addition, as shown in FIG. 8, the BLI signal intensity decreases when 10, 20, and 30 μg of NK-EM is treated for 72 hours in MDA-MB-231 / F, HepG2 / F, and CAL-62 / F cells. I could confirm that.

CCK-8 assay results also showed a similar trend. As shown in FIG. 9, when NK-EM was treated with D54 / F cells and cultured for 48 hours or 72 hours, the cell viability was significantly decreased depending on the treatment concentration. In addition, as shown in Figure 10, MDA-MB-231 / F, HepG2 / F and CAL-62 / F cells also significantly decreased cell viability in a concentration-dependent manner by NK-EM treatment.

The BLI and CCK-8 assay results show that NK-EM is cytotoxic to tumor cells.

3. Tumor cells NK - EM  Confirmation of uptake

In order to confirm the degree of NK-EM absorption of tumor cells, the D54 / F cells were treated with DiD-NK-EM or DiD-NK-Exo for 3 hours, and then stained with D54 / F cells under a confocal laser microscope. Confirmed.

As a result of the confirmation, as shown in FIG. 11, the red stained portion was confirmed, indicating that NK-EM and NK-Exo were absorbed into the cells.

4. NK - From EM FasL  And Perforin  Expression analysis

NK cells display cytotoxicity by releasing cytotoxic effectors, including lytic granules, while at the same time exposing membrane-permeable proteins such as FasL (Fas ligand) to the cell surface, thereby causing cytotoxic substances and target cells. Induces Therefore, it was confirmed that FasL and perforin were present in NK-EM.

As a result of the confirmation, it was confirmed that FasL and perforin exist in both NK-EM and NK-Exo as shown in FIG. 12. As a result of quantitative analysis, it was found that the amount of FasL and perforin was significantly higher in NK-EM compared to NK-Exo ( p <0.001).

In addition, the effect of the FasL inhibitor was confirmed to evaluate whether the cytotoxicity of NK-EM was caused by FasL. As a result, as shown in FIG. 13, it was confirmed that the cell survival rate increased by increasing the BLI signal intensity in the experimental group treated with the FasL inhibitor AF-016 (Kamiya Biomedical Company, USA) ( p <0.001).

5. NK - EM  Anti-tumor effect Mechanism  Confirm

Apoptosis of NK cells consists of two mechanisms: aggregation and perforin / granzymes of the target cell death receptor [FasL or tumornecrosis factor-related apoptosis-inducing ligand (TRAIL) -associated receptor]. The combination of perforin and granzyme induces an intrinsic apoptosis pathway. At the same time, cytochrome-C and death receptor-mediated apoptosis induce an extrinsic apoptosis pathway, resulting in caspase-8, -3 and PARP (Poly (ADP-ribose) polymerase) is activated and the cell is killed.

In order to confirm the mechanism of cell death by NK-EM, it is involved in mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinases (PI3K) signaling pathways associated with apoptosis-associated proteins and cell proliferation. The expression level of the protein was confirmed by Western blot.

As a result, as shown in FIG. 14, it was confirmed that the levels of caspase-3, PARP, and cytochrome-C that were cleaved after 24 hours of NK-EM treatment were increased. The experimental results indicate that NK-EM can induce apoptosis of cancer cells through exogenous and intrinsic apoptosis pathways.

In addition, levels of phosphorylated extracellular signal-regulated kinases (p-ERK) and phosphorylated phosphorylated protein kinase B (p-AKT) phosphorylated AKT decreased 24 hours after NK-EM treatment. The above experimental results indicate that NK-EM can inhibit the proliferation of tumors and has anticancer effects similar to NK cells.

6. In the tumor mouse model NK - EM  Distribution check

D54 / F cells were injected into mice to induce tumor development for 28 days, and distribution of NK-EM in vivo was confirmed 24 hours after DiD-NK-EM administration.

As a result, as shown in FIG. 15A, it was confirmed that tumors were generated in mice injected with D54 / F cells, and as shown in FIG. . On the other hand, in the control group (No tumor) without D54 / F cells, fluorescence signals could not be observed at the same site.

As a result of measuring ex vivo images of organs in vivo in mice, NK-EM was absorbed by confirming fluorescence signals in the spleen, liver, kidney and lungs as shown in FIGS. 16 and 17. there was. Compared to the control group (No tumor), mice injected with D54 / F cells showed weaker fluorescence signals in the liver and spleen (liver: p <0.001, spleen: p <0.05), and NK-EM was present in the tumor tissue. It was confirmed that the absorption ( p <0.001). Through the experimental results, it was confirmed that NK-EM can migrate to tumor cells.

7. In the tumor mouse model NK - EM  Confirm anti-tumor effect

D54 / F cells were injected into mice, and after 10 days, Did-NK-EM was injected to confirm the degree of tumor growth.

As a result, as shown in FIG. 18, it was confirmed that tumor growth was significantly suppressed in the experimental group (NK-EM) administered with NK-EM compared to the control group (PBS) injected with PBS. In addition, as shown in FIG. 19, there was no significant difference in the BLI signal between the control group and the experimental group until the 5th day of Did-NK-EM administration, but from the 9th day of administration, the BLI signal intensity of the experimental group was significantly decreased compared to the control group. Was confirmed ( p <0.001).

Ex vivo BLI showed similar results to in vivo BLI, and as shown in FIG. 20, it was confirmed that the BLI signal was about 4 times stronger in the control group than in the experimental group.

In addition, the ultrasound imaging of the experimental group and the control group (ultrasound imaging) was constructed in a 3-D pattern and quantitatively analyzed. As a result of the analysis, as shown in FIG. 21, it was confirmed that both the volume and the weight of the tumor were significantly decreased in the experimental group compared to the control group. Specifically, the mean tumor volume of the experimental group was 38 mm ³ , and the mean tumor volume of the control group was 170 mm ³ (p <0.001). In addition, as a result of measuring the tumor weight, it was confirmed that the tumor size of the experimental group was 0.122 g per mouse on average, and the control group had a tumor size of 0.45 g per mouse on average.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (7)

As a composition comprising an exosome-like vesicle derived from natural killer cells as an active ingredient,
The exosome-like vesicles include GM-130 (cis-Golgi matrix protein of 130 kD) and cytochrome-C (Cytochrome-C), and are for improving or treating cancer that induces apoptosis of cancer cells. Pharmaceutical composition.
The pharmaceutical composition for improving or treating cancer according to claim 1, wherein the exosome-like vesicle has a size of 50 to 200 nm.
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